Cells traffic molecules to appropriate subcellular organelles and the extracellular environment by the process of vesicle fission from a donor compartment and fusion with an acceptor compartment. A paradigm for vesicle formation, translocation, docking, and fusion has been described and a number of molecules involved in the process have been identified. These include N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAP), and the vesicular- and target-specific SNAP receptors (v- and t-SNAREs, respectively). Many of the signaling pathways providing the cue for vesicle fusion have also been defined. For example, stimulation of chromaffin cells by a cholinergic agonist leads to exocytosis of stored secretory granules and release of catecholamines. Monomeric GTP-binding proteins have been shown to act as regulatory molecules, or "switches", for linking these two processes. The ADP-ribosylation factor (Arf) family of proteins is one such class of regulatory molecule. (Rothman, J. E. and Wieland, F. T. (1996) Science 272: 227-234.)
Arfs were originally identified as cofactors for the cholera toxin-dependent ADP ribosylation of the heterotrimeric G protein, G.sub.S. Arfs were later characterized as monomeric GTP-binding proteins, related structurally to both G protein .alpha.-subunits and Ras proteins. The Arf family members share more than 60% sequence identity, appear to be ubiquitous in eukaryotes, and are highly conserved throughout evolution. For example, Drosophila melanogaster, Shigosaccharomyces pombe, and human ARF1 share more than 90% sequence identity. All Arf family members are myristoylated at the N-terminal glycine residue and associate with membranes in a GTP-dependent manner. Arfs also activate phospholipase D (PLD), a membrane-bound enzyme implicated as an effector of several growth factors. (Boman, A. L. and Kahn, R. A. (1995) Trends Biochem. Sci. 20: 147-150.)
Arf family members fall into three classes according to their size and sequence homology. ARF1, ARF2, and ARF3 form class I, ARF4 and ARF5 form class II, and ARF6 forms class III. These classes occupy different subcellular locations and have been implicated in different transport pathways. Class I Arfs localize to the Golgi where they are involved in the regulation of ER-Golgi and intra-Golgi transport. Class I Arfs are also involved in the recruitment of cytosolic coat proteins to Golgi membranes during the formation of transport vesicles. Class III ARF6 localizes to a tubulovesicular compartment, secretory granules, and the plasma membrane, where it is involved in regulated secretion and recycling. Class II Arfs appear to be cytosolic, but their role has not been elucidated. (Boman, supra; Radhakrishna, H. and Donaldson, J. G. (1997) J. Cell Biol. 139: 49-61; and Tsuchiya, M. (1991) J. Biol. Chem. 266: 2772-2777.)
Arf function is regulated by a GDP-GTP cycle. The role of Arf in vesicle formation has been best studied for ARF1. ARF1 is cytosolic in the GDP bound state, but is associated with membranes when in the GTP bound state. A guanine nucleotide exchange factor (GEF) in the donor compartment recruits ARF1 to the membrane. At the membrane, GTP-ARF1 recruits coat proteins, which assemble together into spherical coats, budding off vesicles in the process. After budding, hydrolysis of bound GTP causes ARF1 to dissociate from the membrane. ARF1 dissociation causes the coat to become unstable and dissociate as well. (Rothman, supra.)
The role of Arf in regulated secretion has been best studied for ARF6. ARF6 is localized to an intracellular compartment in its GDP bound state. This compartment is composed of secretory granules in chromaffin cells and a tubulovesicular compartment in transfected HeLa and CHO cells. ARF6 translocates to the plasma membrane in its GTP bound state and reorganizes the actin skeleton into protrusive plasma membrane extensions, reminiscent of exocytotic events. Analysis of mutants that lock ARF6 into its GDP (T27N) or GTP (Q67L) bound state has verified the nucleotide dependence of this localization pattern. (Radhakrishna, supra.) Studies have shown that this conversion and translocation of ARF6 is regulated by cell signaling pathways. For example, stimulation of chromaffin cells by cholinergic agonists caused ARF6 translocation, and this translocation was required for subsequent catecholamine release. (Galas, M-C. (1997) J. Biol. Chem. 272: 2788-2793.) Together these findings suggest a critical role for ARF6 in regulated secretory processes.
Arf family members have been implicated in several disease processes. Lowe's syndrome, an X-linked disorder characterized by congenital cataracts, renal tubular dysfunction and neurological deficits, may be due to an inability to recruit Arf to the Golgi membrane. (Suchy, S. F. et al. (1995) Hum. Mol. Genet. 4: 2245-2250). It has been suggested that regulation of Arf is also involved in cystic fibrosis, Dent's disease, diabetes and autosomal dominant polycystic kidney disease. (Marshansky, V., et al. (1997) Electrophoresis 18: 2661-2676.)
The discovery of new human ARF-related proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of secretory and epithelial disorders, including those disorders associated with inflammation.